1. Technical Field
The invention relates to methods and materials involved in the rapid detection of mutations.
2. Background Information
Many mutations in human genes exist and are associated with disease or disease risk. For example, a single mutation in exon 10 of the cystic fibrosis gene, delta F508, is associated with cystic fibrosis. In addition, a point mutation in Factor V appears to be associated with thrombosis. Briefly, Factor V is a polypeptide involved in the coagulation cascade. Normally, activated protein C deactivates Factor V to regulate the cascade of coagulation events. One particular mutant form of Factor V, known as Factor V Leiden, contains a single point mutation (G1691A) that renders the polypeptide resistant to activated protein C. Interestingly, Factor V Leiden is found in almost every ethnic group. For example, about 5-7% of the individuals of European or Scandinavian ancestry are carriers. In addition, about 1-2% of the population of other ancestries are carriers. Rather strikingly, over 20% of patients with thrombosis are carriers. Thus, the mutation found in Factor V Leiden may be involved in blood disorders.
Current methods used to detect mutations such as Factor V Leiden (e.g., bidirectional PCR amplification of specific alleles (bi-PASA), or restriction fragment length polymorphism (RFLP)), rely on allele specific primers to amplify DNA that is then analyzed by gel electrophoresis to detect the presence or absence of a specific fragment. The use of gel electrophoresis as a method of detection greatly increases the time required to analyze a sample and limits the number of samples that can be analyzed. Thus, any method that rapidly and accurately detects mutations and does not require gel electrophoresis would be desirable.
The invention involves methods and materials for detecting mutations. Specifically, the invention is based on the discovery of a rapid and reliable detection assay that does not require nucleic acid purification or extraction. The elimination of the need to purify nucleic acid greatly reduces the time needed to perform an assay and does not compromise accuracy. In addition, this simplification of the detection assay reduces the number of steps required for sample preparation and thus reduces the chances of operator error and contamination as well as exposure of the operator to potentially infectious material. Diagnostic kits containing the ingredients needed to perform this simplified mutation detection assay are provided by the invention. Such kits are particularly useful to diagnostic laboratory personnel since the kits can provide all the necessary ingredients in a more uniform manner than otherwise would be available. For example, samples can be analyzed at separate facilities and the results accurately compared to each other since the ingredients in each kit can be prepared in a similar manner by a common vendor. This is particularly important for buffer solutions, oligonucleotide primers, and detection reagents. The invention also provides specific oligonucleotide primers and nucleic acid amplification conditions that allow heterologous amplification products of the human Factor V gene to form stable branching structures. The formation of these stable branching structures is particularly useful for providing a mechanism to detect heterologous alleles verses homozygous alleles. In addition, the invention provides oligonucleotide primers as well as assay conditions that allow for the rapid and accurate screening of multiple samples.
In general, the invention features a composition containing oligonucleotides. The composition amplifies a region of Factor V genomic sequence during a nucleic acid amplification reaction such that amplified products that are heterologous form stable branching structures. In addition, the region contains a potential mutation site. The composition can contain two oligonucleotides that are identical in nucleic acid sequence (e.g., SEQ ID NO:1). Each of the two oligonucleotides can have a different label attached at the 5xe2x80x2 end (e.g., biotin and digoxigenin). Further, the composition can contain two oligonucleotides each having a 5xe2x80x2 portion and a 3xe2x80x2 portion. The 5xe2x80x2 portions of the two oligonucleotides are different in nucleic acid sequence, and the 3xe2x80x2 portions of the two oligonucleotides are identical in nucleic acid sequence. The 3xe2x80x2 portions hybridize to a sequence flanking the mutation site during the nucleic acid amplification reaction. One of the two oligonucleotides can have a nucleic acid sequence as set forth in SEQ ID NO:2, 3, 4, 5, 6, or 7. The amplified products can be less than about 300 bases in length in which case one of the oligonucleotides can have a nucleic acid sequence as set forth in SEQ ID NO:4, 5, 6, or 7. Alternatively, the oligonucleotides of the composition can produce false positive results in a LOCI mutation detection assay less frequently than when Dig-Primer1, Bt-Primer1, Primer2, and Primer3 primers are used in the LOCI mutation detection assay. In this case, the oligonucleotides can include Dig-Primer1, Bt-Primer1, Primer4, and Primer5.
Another aspect of the invention features an oligonucleotide that consists of the sequence as set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.
In another aspect, the invention features a method of detecting heterologous Factor V alleles in an organism. The method includes amplifying a region of Factor V nucleic acid from the organism using oligonucleotides such that amplified products that are heterologous form stable branching structures, and detecting the presence or absence of the stable branching structures. The region contains a potential mutation site and the presence of the stable branching structures is indicative of the presence of the heterologous Factor V alleles in the organism. LOCI can be used for the detecting step. Alternatively, a sandwich immunoassay can be used for the detecting step.
Another embodiment of the invention features a method of detecting homozygous mutant Factor V alleles in an organism. The method includes amplifying a region of Factor V nucleic acid from the organism using oligonucleotides to form amplification products; contacting the amplification products with a solution to form a mixture; and detecting the presence or absence of stable branching structures in the mixture. The region contains a potential mutation site and the solution contains homozygous wild-type Factor V products amplified using the oligonucleotides. The stable branching structures are formed between the amplification products and the homozygous wild-type Factor V products. In addition, the presence of the stable branching structures is indicative of the presence of the homozygous mutant Factor V alleles in the organism. LOCI can be used for the detecting step. Alternatively, a sandwich immunoassay can be used for the detecting step.
Another aspect of the invention features a diagnostic kit for nucleic acid extraction-free detection of heterologous alleles in an organism (e.g., human). The kit contains a cell lysis reagent and two oligonucleotides. The two oligonucleotides are identical in nucleic acid sequence (e.g., SEQ ID NO:1) with each of the two oligonucleotides having a different label attached at the 5xe2x80x2 end (e.g., biotin and digoxigenin). In addition, the two oligonucleotides hybridize to a first region of genomic sequence during a nucleic acid amplification reaction and the first region flanks a potential mutation site. The cell lysis reagent can contain an ingredient that inactivates a polymerase inhibitor. The genomic sequence can contain a Factor V genomic sequence. Alternatively, the genomic sequence can contain a cystic fibrosis genomic sequence. Further, the kit can contain two additional oligonucleotides each having a 3xe2x80x2 portion and a 5xe2x80x2 portion. The 5xe2x80x2 portions are different in nucleic acid sequence, and the 3xe2x80x2 portions are identical in nucleic acid sequence. The 3xe2x80x2 portions hybridize to a second region of the genomic sequence during the nucleic acid amplification reaction, and the second region is located on a side of the potential mutation site that is opposite the side of the first region. The second region can be downstream or upstream of the potential mutation site. One of the two additional oligonucleotides can have a sequence as set forth in SEQ ID NO:2, 3, 4, 5, 6, or 7.
Another aspect of the invention features a method of comparing nucleic acid sequences of alleles in an organism. The method includes amplifying a nucleic acid region of the alleles in a cell lysate to form a cell lysate mixture having amplification products; analyzing the cell lysate mixture for the presence or absence of stable nucleic acid branching structures; and classifying the alleles as heterologous if the stable nucleic acid branching structures are present in the cell lysate mixture.
In another embodiment, the invention features a method of detecting homozygous mutant alleles in an organism. The method includes amplifying a nucleic acid region of the alleles in a cell lysate using oligonucleotides to form a cell lysate mixture having amplification products; contacting the cell lysate mixture with a solution to form a second mixture; analyzing the second mixture for the presence or absence of stable nucleic acid branching structures; and classifying the alleles as homozygous mutant alleles if the stable nucleic acid branching structures are present in the second mixture. The solution contains homozygous wild-type products amplified from homozygous wild-type alleles using the oligonucleotides.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.